1. Field of the Invention
This invention relates to agents and therapy to lessen morbidity and mortality by protecting against septic shock, Adult Respiratory Distress Syndrome (ARDS), and other inflammatory complications of shock. Particularly, this invention relates to the treatment of septic shock and the other complications resulting from septic shock by down-regulating the expression of certain cell-cell adhesion receptors or ligands involved in the inflammatory response during septic shock. More specifically, this invention relates to therapy with antisense oligonucleotides which reduce expression of adhesive proteins and protect against septic shock and reduce associated inflammatory damage (like ARDS). Particularly this invention relates to the use of antisense oligonucleotides complementary to human mRNAs or pre-mRNAs coding for ELAM-1 (Endothelial Leukocyte Adhesion Molecule-1) to be used in a therapeutic treatment of sepsis (henceforth to include sepsis, the sepsis syndrome, septic shock and all other manifestations of the sepsis disease, including but not inclusive of, adult respiratory distress syndrome, multi-organ failure, or cardiovascular dysfunction). Mediators of sepsis produce endothelial dysfunctions that result in the development of an intravascular inflammatory response and subsequent damage to the endothelial cells with migration of leukocytes into the surrounding tissues. This invention also relates to the treatment of sepsis with antisense oligonucleotides targeted to cellular based receptors or their ligands where these receptors or ligands are involved in the inflammatory response during the development of sepsis. This invention further relates to the use of antisense oligonucleotides to inhibit the synthesis of ELAM-1, which is responsible for the adhesion of leukocytes to activated endothelial cells.
2. Description of the Prior Art
Septic shock is defined as a type of shock associated with overwhelming infection. Most commonly, the infection is produced by gram-negative bacteria although other bacteria, viruses, fungi and protozoa may also be causes. As summarized in Infectious Diseases and Medical Microbiology, 2nd edition, edited by Braude et al., Chapter 92, pages 700 et seq.
"Shock is a syndrome of generalized metabolic failure resulting from prolonged inadequacy of tissue perfusion. Its early clinical manifestations reflect malfunction of those organs most dependent on uninterrupted blood flow, particularly the brain, as well as compensatory adjustments designed to maintain adequate arterial pressure. As these adjustments fail, urinary output decreases and biochemical indices of distorted metabolism are detectable; specifically nonoxidative glycolysis with low yield of high energy chemical bonds testifies to the widespread nature of the disorder. In the end, it is the failure of energy production rather than damage to a particular organ that leads to death.
Other terms, such as `circulatory collapse,` `circulatory failure,` and `hypoperfusion,` have been substituted for `shock` in an attempt to pinpoint the specific nature of the derangement. When it occurs as a specific complication of infection, it is referred to as `infectious shock,` `septic shock,` `bacteremic shock,` and even `endotoxin shock.` The last three terms specifically implicate bacterial infection and are therefore too restrictive. Because `infectious shock` is sufficiently broad as well as concise, this term will be used in the present chapter.
Shock may occur in the course of almost any severe infection, but it is particularly characteristic of bacteremia due to gram-negative bacilli. . . . The importance of endotoxin, the lipopolysaccharide (LPS) composing part of all gram-negative cell walls, is readily apparent because it produces a similar syndrome in experimental animals. Partly because of the extensive use of endotoxin as an investigative tool, endotoxin shock is commonly regarded as the prototype of infectious shock."
The shock is believed to be caused by the action of endotoxins, other products of the infectious agent, or host mediators released in response to the infectious agent on the vascular system. Such action causes altered patterns of perfusion of tissues and large volumes of blood to be sequestered in the capillaries and veins.
Sepsis, the sepsis syndrome, and septic shock are not discrete entities, but rather terms that delineate increasingly severe stages of the same disease. Septic shock, a frequently fatal reaction following bacterial infection, has been estimated to occur at a rate of 175 per 100,000 people yearly in the general population and rises to 500 per 100,000 for those people admitted to hospitals (Johnston, J. (1991) J. NIH 3: 61-65). Estimates range up to 400,000 cases of sepsis, 200,000 bouts of septic shock, and 100,000 deaths annually in the United States due to the septic shock induced syndrome (Snell, J. and J. E. Parrillo (1991) Chest 99: 1000-1009). Up to 40-50% of patients who develop septic shock die. The manifestation of septic shock involves a severe decrease in systemic vascular resistance and maldistribution of blood flow. Septicemia, a systemic disease associated with the presence and persistence of pathogenic microorganisms or their toxins in the blood, is currently ranked as the thirteenth leading cause of death in the United States (Annual Summary of Births, Marriages, Divorces, and Deaths: United States, 1988. Hyattsville, Md.: U.S. Department Health and Human Services, Public Health Service, CDC, 1989: 7. Monthly vital statistics report. 1989: 37[13]). Reasons underlying this high incidence of death from septic shock involve increased usage of cytotoxic and immunosuppressive drug therapies which impairs host defense mechanisms or increased use of invasive diagnostic devices or increased patient age (Snell, J. and J.E. Parrillo (1991) Chest 99: 1000-1009). Further causes of impaired host defense mechanisms include diabetes, malignant neoplasms, cirrhosis or extensive burns. The rising rate of infections from organisms other than gram-negative bacteria also contribute to the rise in septic shock induced death. Any bacteria can cause septic shock, however, the gram-negative bacteria (E. coli, Pseudomonas sp. and Bacteroides sp. ) in particular evoke septic shock due to the presence of lipopolysaccharide (LPS) in their cell walls. Bacterial LPS, also known as endotoxin, at concentrations as low as a few .mu. g/L can activate immune cells. The majority of damage induced from the presence of LPS is not due to the actual LPS itself, but is in fact a result of the body's complex reaction to the foreign LPS. This response is mediated by immune cell activation and the resultant damage that these activated cells cause to the host tissues.
Septicemia is difficult to reverse and the preferred treatment following the initial signs of hypoperfusion or shock include infusion of normal saline or lactated Ringer's solution. If shock persists then an aggressive fluid challenge is begun and the use of dopamine and/or norepinephrine is recommended. Cardiovascular insufficiency results from alterations to the myocardium and the vasculature and it is myocardial dysfunction that is responsible for hypotension or multiple organ system failure (Snell, J. and J. E. Parrillo (1991). Chest 99: 1000-1009). Unresponsive hypotension usually results from low systemic vascular resistance due to cardiovascular insufficiency which can not be corrected by any therapy. Multiple organ failure usually affects the lungs, kidneys, liver, heart, and central nervous system.
Treatment of septic shock is complex, requiring therapies directed at ameliorating the source of infection [antibiotics], blocking effects of products of the infectious agent and inflammatory mediators on tissues [anti-endotoxin (patent Young et al. U.S. Pat. No. 4,918,163) and anti-cytokine agents (patent Mandell et al. U.S. Pat. No. 4,965,271)], and maintenance of cardiovascular function [volume expansion and pressor agents]. However, mortality still runs at about 100,000 patients per year (40 to 50% of those in shock) and no therapies are available to prevent vascular contractile defects.
Other current approaches to the treatment of sepsis or septic shock involve neutralization of LPS with specific monoclonal antibodies, interference of cytokine-mediated immune responses, or inactivation of cell adhesion proteins with monoclonal antibodies. Targeting of LPS mediated sepsis, however, will be effective only against gram-negative bacteria since LPS is only found in their cell walls. Monoclonal antibodies to the lipid A domain of LPS have had some success at intervention with LPS mediated septic shock from gram-negative bacteria, but not for non-gram-negative induced septic shock (Ziegler, E.J et al. (1991) N. Eng. J. Med. 324: 429-436). Thus, while the gram-negative LPS may be the most potent inducer of sepsis, gram-positive bacterial infections occur in 60-70% of all cases. Intervention with cytokine mediated activation of the immune response as a means of preventing septic shock would not only interfere with gram-negative induced sepsis, but also shock caused by gram-positive bacterial infection or other agents. The development of an effective therapy to treat all bacterial induced septic shock would be of obvious benefit to patients who are at an increased risk of bacterial induced sepsis and provide increased survival from septic shock and the complications that arise during septic shock induced dysfunctions. Another approach would be interference with the cellular response to the various endogenous mediators (cytokines, PAF, arachidonic acid metabolites, histamine, endorphins, etc) responsible for vasculature effects. These approaches are not currently approved for therapy and are in clinical trials.
One of the major effects experienced by the vasculature is destruction of endothelial cells by leukocytes. Inflammation is characterized by the local accumulation of leukocytes, plasma proteins, and fluid usually at an extravascular site. Inflammatory processes are intrinsically destructive to the surrounding tissues and may, in certain circumstances such as allograft rejection or sepsis, be more harmful than beneficial. Thus, an appropriate strategy for the treatment or prevention of sepsis or septic shock would be down-regulation, but not total ablation, of the inflammatory response. Down-regulation of specific cell adhesion receptors and/or ligands to the receptors would be one approach to preventing, or lessening, the inflammatory mediated damage to endothelial cells in the vasculature.
The involvement of the immune response in the development of septic shock and its lethal consequences provides a target that is applicable to the use of antisense oligonucleotides. Antisense oligonucleotides can be used to inhibit expression of the key receptors and cellular ligands involved in the activation of the immune response. The migration of leukocytes into tissues is the central event in the immune or inflammation response. This migration to and subsequent emigration into the tissue is responsible for the successful host response to injury and infection. The leukocytes are also potentially harmful and contribute to the pathology of many inflammatory disorders. The precise mechanism of this injury is not known, but the generation of free oxygen radicals and release of proteolytic enzymes have been implicated and may act together in leukocyte induced endothelial cell damage (Varani, J. et al. (1989), Am. J. Path. 135: 429-436). Evidence for the leukocyte adhesion to endothelial cells has been attributed to specific surface proteins. Involved in the binding of leukocytes to activated endothelium is a family of endothelial cell adhesion molecules known as the selectins or LECCAMs. One member of this family is the endothelial leukocyte adhesion molecule-1, also known as ELAM-1. ELAM-1 is a 110 kD cell-surface glycoprotein of endothelial cells that binds neutrophils and perhaps monocytes (Bevilacqua, M.P et al. (1987) Proc. Natl. Acad. Sci. USA 84: 9238-9242). There are two pathways for the adhesion of leukocytes to endothelium: 1 ) an immediate adhesion that is not dependent upon the de novo synthesis of proteins, and 2) a delayed adhesion (1-2 hours) that is dependent upon the synthesis of proteins (Osborn, L. (1990) Cell 62: 3-6). The synthesis and surface presentation of ELAM-1 in endothelial cells suggests that ELAM-1 may be involved in the second, or delayed, component of leukocyte adhesion (Bevilacqua, J.S. et al. (1987) Proc. Natl. Acad. Sci. USA 84:9238-9242 and Bevilacqua, J.S. et al. (1989) Science 243: 1160-1165). Endotoxin or LPS can increase the adhesion of leukocytes to endothelial cells through the biosynthesis and expression of ELAM-1. Stimulation of human umbilical vein endothelial cells (HUVECs) with either interleukin-1 or tumor necrosis factor-alpha results in more than a 100-fold increase in the surface presentation of ELAM-1 (Osborn, L. et al. (1990) Cell 62: 3-6). Also, the stimulation of ELAM-1 synthesis and its presentation on the surface of HUVECs has shown to be mediated with endotoxin (Munro, J.M. et al. (1991) Lab. Invest. 64: 295-299). Injection of endotoxin into the skin of baboons results in the strong, widespread endothelial binding of anti-ELAM-1 antibodies in the venules by 2 hours after the injection. This two hour delay in presentation of ELAM-1 to the venules correlated with the time course for adherence of neutrophils. After 9 hours the expression of ELAM-1 and the ability of ELAM-1 to be recognized by the antibodies had declined to pre-injection levels (Munro, J.M. et al. IBID). Thus, this study demonstrates that the early dermal accumulation of neutrophils after injection of endotoxin is associated with the endothelial cell expression of ELAM-1. These in vivo effects closely parallel the in vitro evidence concerning the induction of ELAM-1 by endotoxin and the role of ELAM-1 in neutrophil adhesion. The ability of antibodies to ELAM-1 to block the adhesion of neutrophils, eosinophils, and basophils induced in vitro in HUVECs by interleukin-1 suggests that ELAM-1 plays an important role in the requirement of these cells during the inflammatory response (Bochner, B.S. et al. (1991) J. Exp. Med. 173:1553-1556 and Carlos, T. et al. (1991) Blood 77: 2266-2271).
Leukocytes, especially neutrophils, may injure endothelial cells (Pober, J.S. and Cotran, R.S. (1990) Transplant. 50: 537-544). While the exact mechanism is not known, the damage may be induced by oxygen radicals or proteolytic enzymes released from the neutrophils. Damage to the endothelial cells lining the vasculature results in leakage into the surrounding tissues. The pulmonary leak that is produced in adult respiratory distress syndrome, a resultant complication of septic shock, most often results from neutrophilmediated capillary injury (Helfin, A.C. and K.L. Brigham (1981) J. Clin. Inv. 68: 1253-1260).
ELAM-1 eDNA and the genomic clones have been isolated and the nucleic acid sequence of the pre- and mature-mRNA can be determined from these sequences (Goelz, S.E et al. (1990) Cell 63: 1349-1355; Hession, C. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1673-1677; and Collins, T. et al. (1991) J. Biol. Chem. 266: 2466-2473). Thus, the ability to target the pre- or mature-mRNA with antisense oligonucleotides with the express purpose of down-regulating the synthesis of ELAM-1 is immediately possible.
Involved in the activation of the inflammatory and immune response, as during the development of sepsis and septic shock, is the expression of many essential cell adhesion proteins and receptors. Adhesion molecules are activated by various cellular mediators, exogenous or endogenous to the host, and therefore, the logical approach is down-regulation of adhesion protein expression as opposed to treatments aimed at the multiple activators. Thus, the use of antisense oligonucleotides to specifically down-regulate adhesion protein expression would be of obvious advantage to most therapeutic approaches to septic shock.
Research by others into PKC inhibition and treatment of inflammatory responses have disclosed that endothelial cells express adhesive proteins in response to sepsis associated stimuli such as endotoxin or cytokines, such as interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF). Magnuson, D.K. et aL ((1989) Surgery 106: 216-223) and Lane, T.A. et al. ((1990) Biophys. Res. Comm. 172: 1273-1281) have shown that these adhesive proteins can be reduced on endothelial cell surfaces by inhibition of PKC with staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7). Surface presentation of these adhesive proteins enhances white blood cell infiltration and activation which can result in tissue damage in inflammatory states like septic shock. In addition, PKC activation enhances endothelial cell permeability resulting in edema. This response to inflammatory agents was also abrogated by exposure of the cells to the PKC inhibitor H7 (Lynch, J.J. et al. (1990) J. Clin. Invest: 85:1991-1998). Abnormal leukocyte accumulation is implicated in a variety of inflammatory states such as: reperfusion injury, autoimmune diseases, and acute respiratory distress syndrome (ARDS). The damage is thought to result from the release of toxic oxygen radicals and proteases that potentiate tissue damage. The use of anti-adhesive protein antibodies or adhesive like proteins was shown to reduce tissue damage in select models of reperfusion injury (Vedder, N.B. et al. (1988), J. Clin. Inv. 81: 939-944; P.J. Simpson et al. (1988) J. Clin. Inv. 81: 624-629; M. J. Horgan et al. (1989) Am. J. Physiol. 259: L315-319; International Patent Application of inventors Vadas, M, and M. Berndt (1991) Application #WO 91/07993) and endotoxin induced damage (H. Rosen and S. Gordon (1989) Br. J. Exp. Path 70: 385-394). When antibodies are used as a treatment they do not control the levels of expression of these proteins and the antibodies typically have short half-lives in circulation. An additional complication of antibodies is the potential for immunogenic reactions to large foreign proteins. Compared to antibodies, smaller molecules like antisense oligonucleotides can overcome these disadvantages and also provide selective control of expression of a single cellular protein.
The mRNA coding for ELAM-1 has been cloned and the nucleic acid sequence is available for selective targeting with antisense oligonucleotides (Collins, T. et al. (1991) J. Biol. Chem. 266: 2466-2473).